Limited tolerance towards folded elements during secretion of the autotransporter Hbp

Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a β-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane

A revision of Vernicomacanthus Miles with comments on the characters of stem-group chondrichthyans

International audiencehe ‘acanthodian’ fishes provide key anatomical insights into the deepest branches of the chondrichthyan stem group. We review the anatomy of the acanthodian Vernicomacanthus uncinatus from the Lochkovian (Lower Devonian, 419.2–410.8 Ma) of Scotland based on eight articulated fossils, one of which is newly described. Broadly, the anatomy of V. uncinatus fits with that of contemporaneous acanthodians such as Climatius and Parexus, with a head covered by robust tesserae, an enlarged postorbital scale, an armoured shoulder girdle, and many pairs of ventrolateral spines. However, it departs from this anatomy in key respects. Its pectoral fin spines are obliquely ridged and posteriorly denticulated, similarly to Carboniferous gyracanth stem-group chondrichthyans. Its scales consist of multiple anteroposteriorly aligned odontodes, similarly to many Palaeozoic ‘sharks’. And its endoskeletal shoulder girdle may have a posterolateral angle, previously observed only in shark-like chondrichthyans. We propose that the differences between V. uncinatus and its congeneric, V. waynensis, which include potentially phylogenetically significant characters of the shoulder girdle and spines, are sufficient to erect a new genus for V. waynensis:Dobunnacanthus gen. nov. The scales of Vernicomacanthus are identical to those of the ‘shark’ scale genus Altholepis, suggesting that some such scales may instead belong to taxa with acanthodian-like gross anatomies. Based on these scales we highlight potential patterns in chondrichthyan scale evolution, in particular the axial addition of odontodes. Anatomical similarities between Vernicomacanthus and gyracanths, highlighted by previous authors, may indicate the existence of a grade including these and similar acanthodian-grade taxa placed relatively crownwards in the chondrichthyan stem-grou

The Use of a Stringent Selection System Allows the Identification of DNA Elements that Augment Gene Expression

The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner

Characterization of ftsZ Mutations that Render Bacillus subtilis Resistant to MinC

Background: Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites. Methodology/Principal Findings: We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro. Conclusions/Significance: Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.

Overexpression of Mineralocorticoid Receptors in the Mouse Forebrain Partly Alleviates the Effects of Chronic Early Life Stress on Spatial Memory, Neurogenesis and Synaptic Function in the Dentate Gyrus

Evidence from human studies suggests that high expression of brain mineralocorticoid receptors (MR) may promote resilience against negative consequences of stress exposure, including childhood trauma. We examined, in mice, whether brain MR overexpression can alleviate the effects of chronic early life stress (ELS) on contextual memory formation under low and high stress conditions, and neurogenesis and synaptic function of dentate gyrus granular cells. Male mice were exposed to ELS by housing the dam with limited nesting and bedding material from postnatal day (PND) 2 to 9. We investigated the moderating role of MRs by using forebrain-specific transgenic MR overexpression (MR-tg) mice. Low-stress contextual (i.e., object relocation) memory formation was hampered by ELS in wildtype but not MR-tg mice. Anxiety like behavior and high-stress contextual (i.e., fear) memory formation were unaffected by ELS and/or MR expression level. At the cellular level, an interaction effect was observed between ELS and MR overexpression on the number of doublecortin-positive cells, with a significant difference between the wildtype ELS and MR-tg ELS groups. No interaction was found regarding Ki-67 and BrdU staining. A significant interaction between ELS and MR expression was further observed with regard to mEPSCs and mIPSC frequency. The ratio of evoked EPSC/IPSC or NMDA/AMPA responses was unaffected. Overall, these results suggest that ELS affects contextual memory formation under low stress conditions as well as neurogenesis and synaptic transmission in dentate granule cells, an effect that can be alleviated by MR-overexpression

Structure and Function of the Shoulder Girdle in Dipnoans: New Material from Dipterus valenciennesi

The pectoral girdle, including the "anocleithrum", of Dipterus valenciennesi is described from Caithness, Scotland. This makes it the oldest known dipnoan genus for which the "anocleithrum" is known. The details of the cleithrum, clavicle and scapulocoracoid, are described. The median part of the "anocleithrum" has a pair of long processes for attachment to the endocranial process posterior to bone "I". Problems of the homology of the "anocleithrum" are discussed. The hyomandibular of Chirodipterus australis is used as an example of the Devonian dipnoans. The structure of the opercular and its muscles for the attachment of the hyomandibular process, suggests that the hyomandibular / opercular arrangement was the same kind as in Dipterus valenciennesi. The rotation of the hyomandibular in opening and closing the operculum, makes for a distinctive means for controlling the ventilation of the gill chambers. A comparison of the "anocleithrum" with those of other Palaeozoic dipnoans is presented, and the function of the dipnoan "anocleithrum " is outlined. In dipnoans the gill arches have neither hypobranchials nor pharyngobranchials. We consider that the presence of the "anocleithrum", the movement of the hyomandibular in moving the opercular, and the restriction of the gills, is intimately related. The introduction of these features results from the modification by gene regulation near the beginning of the Devonian. The restriction of these features to the Dipnoi provides a distinctive way to identify the members ofthat group

Non-hydrolysable GTP-γ-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP-dependent way. GTP, but not guanosine-5'-O-(3-thiotriphosphate) (GTP-γ-S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non-hydrolysable analogue GTP-γ-S bind FtsZ with similar affinity, whereas the non-hydrolysable analogue guanylyl-imidodiphosphate (GMP-PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP-γ-S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP-γ-S exchanges only a small portion of the FtsZ polymer-bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP-containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.

Data from: The oldest actinopterygian highlights the cryptic early history of the hyperdiverse ray-finned fishes

Osteichthyans comprise two divisions, each containing over 32,000 living species: Sarcopterygii (lobe-finned fishes and tetrapods) and Actinopterygii (ray-finned fishes). Recent discoveries from China highlight the morphological disparity of early sarcopterygians and extend their origin into the late Silurian. By contrast, the oldest unambiguous actinopterygians are roughly 30 million years younger, leaving a long temporal gap populated by fragments and rare body fossils of controversial phylogenetic placement. Here we reinvestigate the enigmatic osteichthyan Meemannia from the Early Devonian (∼415 million years ago) of China, previously identified as an exceptionally primitive lobe-finned fish. Meemannia combines “cosmine”-like tissues taken as evidence of sarcopterygian affinity with actinopterygian-like skull roof and braincase geometry, including endoskeletal enclosure of the spiracle and a lateral cranial canal. We report comparable histological structures in undoubted ray-finned fishes and conclude that they are general osteichthyan features. Phylogenetic analysis places Meemannia as an early-diverging ray-finned fish, resolving it as the sister lineage of Cheirolepis plus all younger actinopterygians. This brings the first appearance of ray-fins more in line with that of lobe-fins and fills a conspicuous faunal gap in the otherwise diverse late Silurian-earliest Devonian vertebrate faunas of the South China Block